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Journal of Chromatography B, 863 (2008) 115–122

Validation of an HPLC–MS/MS method for the simultaneous determination

of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl
mercapturic acid in urine as biomarkers of
exposure to benzene, toluene and xylenes
Laura Sabatini ∗ , Anna Barbieri, Paolo Indiveri,
Stefano Mattioli, Francesco Saverio Violante
Occupational Medicine Unit, University of Bologna, S. Orsola-Malpighi Hospital, via Palagi 9, 40138 Bologna, Italy
Received 26 October 2007; accepted 11 January 2008
Available online 18 January 2008

Abstract
A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and fully validated, according to U.S. Food and Drug
Administration guidance, for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic
acid in human urine as biomarkers of exposure to benzene, toluene and xylenes (BTX). After solid phase extraction and LC separation, samples
were analyzed by a triple–quadrupole mass spectrometer operated in negative ion mode, using isotope-labeled analogs as internal standards (ISs).
The method meets all the validation criteria required. The limits of detection of the three analytes, ranging from 0.30 to 0.40 ␮g l−1 , and the high
throughput make the method suitable for the routine biological monitoring of co-exposure to BTX both in the occupational and environmental
settings. The validated method was applied to assess exposure to BTX in a group of 354 urban traffic wardens.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Liquid chromatography–mass spectrometry; Mercapturic acids; Benzene; Toluene; Xylenes

1. Introduction Benzene is classified from the International Agency for

Aromatic hydrocarbons benzene, toluene and isomeric
ortho-, meta- and para-xylene (BTX) are important industrial
chemicals widely used, singly and in combination, as organic
solvents and in the synthesis of other chemicals. In addition,
these compounds are volatile components of gasoline and con-
stituents of tobacco smoke. Due to their formation in many
combustion processes, they are widespread environmental pol-
lutants [1]. Thus, the general population undergoes lifelong
exposure to these pollutants, and some categories of workers,
like gas station attendants and oil refinery workers, are exposed
at particularly high levels.
∗ Corresponding author at: Unità Operativa di Medicina del Lavoro, Univer-
sità di Bologna, via Palagi 9, 40138 Bologna, Italy. Tel.: +39 051 4290216;
fax: +39 051 301968.
E-mail address: l.sabatini@unibo.it (L. Sabatini).
Research on Cancer (IARC) [2] as a human carcinogen (group
1). Currently, no evidence exists to suggest that toluene and
xylenes are carcinogenic. Nevertheless, exposure to high con-
centrations of these compounds can induce changes in the
central nervous system and other neurotoxic effects [3–5].
The metabolism of these aromatic compounds has been thor-
oughly investigated [6–8]. At least 90% of the absorbed BTX
is excreted through the kidneys as metabolites, including
trans,trans-muconic acid and phenol for benzene, hippuric acid
and o-cresol for toluene and methylhippuric acids for xylenes.
However, the validity of an exposure biomarker mainly relies
on its specificity for the toxic compound under consideration.
Among the known urinary metabolites, mercapturic acids (MAs)
have been recently considered the most specific biomarkers
of aromatic compounds, despite their relatively low levels of
metabolic production (1% or less of the absorbed BTX) [8,9].
MAs are products of a metabolic detoxification pathway and are
excreted in urine after the reaction of electrophilic intermediates

1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2008.01.022
116 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122
with endogenous glutathione [10]. At low levels of exposure,
phenylmercapturic acid (PMA) has indeed been validated as
a more specific biomarker for benzene [7,11]. Furthermore,
benzylmercapturic acid (BMA) and o-methylbenzyl mercap-
turic acid (MBMA) have been proposed as reliable biomarkers
of exposure to toluene [12] and xylenes [13], respectively.
In order to perform biological monitoring of occupationally
and/or environmentally exposed individuals, several analytical
methods have been developed for the determination of MAs
in urine, based on high performance liquid chromatography
(HPLC) or gas chromatography–mass spectrometry (GC–MS)
[14]. Among published methods, HPLC/tandem mass spectrom-
etry (MS/MS) is currently considered as the first choice for MAs
determination in human urine due to its high specificity and sen-
sitivity. Several HPLC–MS/MS methods have been reported for
the determination of PMA and BMA in urine [13,15–20] and
Gonzalez-Reche et al. [21] described a method for the quantifi-
cation of dimethylphenyl mercapturic acid (a MA derived from
the metabolism of o-xylene) in urine as a biomarker of exposure
to xylenes. However, the metabolism of alkyl aromatics mainly
occurs at the side chain [22–24], producing, in the case of xylene,
a higher amount of MBMA. Nevertheless, to our knowledge,
no HPLC–MS/MS methods have been developed for the anal-
ysis of MBMA in urine. In addition, analytical methods for the
simultaneous determination of several MAs would be particu-
larly relevant in cases of co-exposure to several solvents. In fact,
many studies have demonstrated that administration of toluene
and xylenes to rats causes depletion of glutathione in the liver,
influencing the metabolic routes of these and other toxicants
[6,25]. Moreover, it is important to note that none of the above-
mentioned methods was fully and rigorously validated following
specific guidelines. As a consequence, little data are available
concerning the reliability of previously obtained results.
The aim of our study was to validate an HPLC–MS/MS
method to simultaneously determine PMA, BMA and MBMA
in human urine for a complete and accurate monitoring of expo-
sure to BTX. For this purpose, a 96-well solid phase extraction
(SPE) procedure and a micro(␮)-HPLC separation coupled to
electrospray ionization (ESI)-MS/MS were used. This analyti-
cal method was fully validated according to the FDA [26], using
two deuterated analogs as internal standards (ISs) and different
urine lots to assess matrix effect variability. Moreover, the vali-
dated method was successfully applied to measure exposure to
BTX in a group of 354 traffic wardens.
2. Experimental
2.1. Reagents and chemicals
All chemicals were of analytical reagent grade. Formic acid
(98%) was purchased from Fluka (Buchs, Switzerland) and
methanol of chromatographic grade was obtained from Merk
(Darmstadt, Germany). Deionized water (18.2 M cm) was pro-
duced with a Direct-Q Millipore Waters system (Millford, MA,
USA). Isolute® ENV+ cartridges (50 mg, 1 ml) and Isolute®
C18 cartridges (50 mg, 1 ml) were purchased from IST (Mid
Glamorgan, UK); EvoluteTM ABN cartridges (25 mg, 1 ml) were
purchased from Argonaut Technologies Ltd. (Mid Glamorgan,
UK). PMA, BMA and MBMA were supplied by Tokyo Kasei
(Prodotti Gianni, Milan, Italy). Deuterated analogs PMAd-5 and
BMAd-7 were custom synthesized by Alchemy s.r.l. (Bologna,
Italy) to use as internal standards (ISs). All the standard com-
pounds were of the highest available purity (>99%). Stock
solutions were prepared by dissolving pure powder of each ana-
lyte and ISs in methanol to give a concentration of 200 mg l−1 .
Stock solutions were stored at −20 ◦ C. Working standard solu-
tions (500 ␮g l−1 for PMAd-5 and BMAd-7; 50–1000 ␮g l−1 for
PMA, BMA and MBMA) were prepared weekly by dilution of
the stock solutions in MeOH/20 mM formic acid 1:1 (v/v) and
were kept at +4 ◦ C.
2.2. Apparatus
HPLC–ESI-MS/MS analysis was performed using a series
1100 ␮-HPLC system (Agilent Technologies Inc., Waldbronn,
Germany), equipped with a thermostatted well-plate autosam-
pler and thermostatted column compartment modules. The
HPLC system was interfaced to an API 2000 triple–quadrupole
mass spectrometer from PE Sciex (Concord, ON, Canada)
equipped with a TurboIonSprayTM source. A nitrogen generator
system 75-72 Whatman (Maidstone, Kent, UK) was employed
to produce N2 , used as curtain and auxiliary gas. Instrument
control and data acquisition were performed with Analyst Soft-
ware PE Sciex (rev. 1.3.2). An EZ-2plus Evaporator (GeneVac
Ltd., Ipswich, UK) was used for solvent evaporation. SPE was
performed on a vacuum system 96-well plated VacMaster (IST).
2.3. Extraction procedure
Urine samples were collected and stored at −20 ◦ C. Prior to
analysis, each sample was thawed, vigorously mixed and then
centrifuged at 1500 × g for 10 min to obtain clear supernatant.
The clean-up procedure was optimized by comparing three dif-
ferent types of reversed phase SPE cartridges: Isolute® C18 ,
Isolute® Env+ and EvoluteTM ABN.
EvoluteTM ABN cartridges were finally chosen for their
higher recovery (data not shown). We loaded 1 ml of centrifuged
and diluted (1:1 with aqueous formic acid 1%, v/v) urine sam-
ple spiked with 5 ␮l of each IS working solution. The cartridges
were previously conditioned with 1 ml of MeOH and 1 ml of
aqueous formic acid 0.1% (v/v). The stationary phase was then
washed with 1 ml of aqueous MeOH solution (10%, v/v) and
the analytes were eluted with two aliquots (250 ␮l) of methanol.
The eluate was evaporated to dryness under vacuum at +40 ◦ C in
an EZ-2 Plus concentrator. The residue was redissolved in 50 ␮l
of 20 mM formic acid/MeOH 1:1 (v/v) and a volume of 0.5 ␮l
was injected into the ␮-HPLC system.
2.4. Liquid chromatography
HPLC analysis was performed at a flow rate of 10 ␮l min−1
using 20 mM formic acid (solvent A) and methanol (solvent B).
Separation was accomplished on a Synergi 4u Max-RP capillary
column (0.5 mm × 50 mm, 4 ␮m, 80 Å, Phenomenex® Torrance,
 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122
Fig. 1. Product ion mass spectra (ESI− ) and ion structures of: (A) PMA (precur-
sor ion m/z = 238); (B) BMA (precursor ion m/z = 252); (C) MBMA (precursor
ion m/z = 266).
CA, USA), with an appropriate pre-column (Synergi 4u Max-
RP, 0.5 mm × 20 mm, 4 ␮m, 80 Å, Phenomenex® ). The elution
gradient was performed as follows: after 1 min of isocratic elu-
tion (solvent A 65%, solvent B 35%), solvent B was increased
from 35 to 90% in 2 min along a linear gradient curve. The iso-
cratic elution was held for 12 min, after which solvent B was
decreased from 90 to 35% in 1 min and column equilibration
was conducted isocratically for 11 min (total run time 27 min).
The autosampler tray was thermostatted at +15 ◦ C.
2.5. Mass spectrometry
MS/MS analysis was performed on a triple–quadrupole mass
spectrometer operated in negative ion mode. Polypropylene
glycol standard solution was used to calibrate the instrument
over the m/z range 100–900 and to adjust the resolution to
0.7 m/z for MS/MS analysis. Product ion mass spectra from
the deprotonated molecular ions [M − H]− were recorded in
the range 100–300 m/z for analytes (PMA, BMA and MBMA)
and ISs (PMAd-5 and BMAd-7) in order to characterize the
fragmentation behavior of each compound. Fig. 1 shows the
product ion mass spectra of PMA, BMA and MBMA. Tran-
sitions of the deprotonated molecular ions [M − H]− for the
117
analytes and ISs were monitored for quantitative analysis in
multiple reaction monitoring (MRM) mode (dwell time/channel
600 ms, unitary resolution). Transitions 238 → 109, 252 → 123,
266 → 137, 243 → 114 and 259 → 130 were selected for PMA,
BMA, MBMA, PMAd-5 and BMAd-7, respectively. Instrumen-
tal parameters were optimized for each analyte by infusion of the
corresponding standard solution (0.5 mg l−1 in 20 mM formic
acid/MeOH 1:1, v/v) at a flow rate of 10 ␮l min−1 , using a
syringe pump integrated in the API 2000 mass spectrometer.
Nitrogen was used as curtain and auxiliary gas and air was
used as nebulizer gas. Electrospray conditions for PMA, BMA,
MBMA and ISs were curtain gas, 20 psi; ion-spray voltage,
−4500 V; nebulizer and auxiliary gas, 40 and 60 psi, respec-
tively; turbo temperature, 250 ◦ C; collision energy, −18.0 eV;
declustering potential, −10 V; focusing and entrance potentials,
−350 and −8 V, respectively.
2.6. Urine specimens
For method validation purposes, spot urine samples were col-
lected from healthy non-smoker male and female subjects not
occupationally exposed to any organic solvents. However, since
in urban areas BTX are ubiquitous pollutants, it is extremely
difficult to acquire real blank urine samples. Due to the unavail-
ability of a blank urine matrix, a screening procedure was used
to determine which urine samples contained the lowest concen-
trations of PMA, BMA and MBMA. Thirty lots of urine were
analyzed by HPLC–MS/MS in order to quantify the background
concentrations of the three analytes. The creatinine concentra-
tion of each urine sample was also measured using the Jaffé
method [27].
Six lots with urinary creatinine ranging between 0.3 and
3.0 g l−1 were then chosen for their low levels of MAs (PMA
and MBMA: non-detectable; BMA: between 1.0 and 3.2 ␮g l−1 ,
mean: 1.7 ␮g l−1 ). These urine samples were used separately to
assess matrix effect and pooled to obtain calibration and quality
control (QC) samples.
2.7. Statistics
A skewness–kurtosis test was used to assess the normal dis-
tribution of values. In case of normal distributions, continuous
variables were tested by the Student’s test. For non-normal dis-
tributions, the Wilcoxon rank–sum test was used. Stata 8.0 SE
software (Stata Corporation, TX, USA) was used for all analy-
ses, with significance set at p < 0.05.
2.8. Calibration curves
Calibration samples were prepared in triplicate in urine, in
accordance with the FDA requirements. Pooled urine was spiked
with working standard solutions of the three analytes, in order to
obtain seven concentration levels (0, 0.6, 1, 2, 10, 25, 50 ␮g l−1
for PMA; 0, 0.7, 1, 2, 10, 25, 50 ␮g l−1 for BMA and 0, 0.8,
1, 2, 10, 25, 50 ␮g l−1 for MBMA). The zero sample was a
pooled urine sample (matrix) with ISs. Calibration samples were
extracted by SPE and analyzed by ␮-HPLC–ESI-MS/MS (as
118 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122
described above) in the same day and every 50 samples. Ana-
lyte peak area/IS peak area ratios were plotted against nominal
concentrations (PMAd-5 was used as IS for PMA and BMAd-
7 was used for BMA and MBMA). Concentrations were back
calculated from the corresponding calibration curves.
2.9. Validation procedures
The method was fully validated according to the FDA guide-
lines. Limit of detection (LOD), limit of quantification (LOQ),
linearity, precision and accuracy, recovery, ion suppression,
specificity and stability of the HPLC–ESI-MS/MS method were
determined.
2.9.1. Limit of detection and limit of quantification
The LOD and LOQ of the method were assessed for each ana-
lyte by sextuplicate analysis of the calibration samples. The LOD
and LOQ were estimated by calculating the standard error of the
intercept (Sb) on the calibration curves (y = mx + b). For BMA,
the calibration curve was constructed by subtracting the back-
ground level peak area from the values determined for the spiked
specimens. The LOD and LOQ were expressed as two and four
times the Sb/m, respectively. Precision and accuracy at the LOQ
level should meet the FDA acceptance criteria (R.S.D. ≤ 20%
and bias ≤20%).
2.9.2. Precision and accuracy
The precision and accuracy of the entire method were
assessed for each analyte at three QC concentration levels in
pooled urine samples spiked at 2, 20 and 50 ␮g l−1 . QCs were
extracted and analyzed in six replicates on the same day (intra-
day precision and accuracy) and on six different days within
2 months (inter-day precision and accuracy). Precision was
expressed as the relative standard deviation (R.S.D.) and accu-
racy was calculated as the relative difference between measured
and nominal concentration of the QC samples (bias%).
2.9.3. Recovery and ion suppression
SPE recovery was calculated for the three analytes by
comparing the area responses of extracted and non-extracted
standard solutions containing PMA, BMA and MBMA at
three concentrations (2, 20 and 50 ␮g l−1 ), each analyzed in
triplicate.
Moreover, ion suppression was evaluated according to the
FDA guidelines using two different procedures. Six differ-
ent human urine samples were processed (as described above)
and dry extracts were dissolved with 50 ␮l of working solu-
tion at 10 ␮g l−1 for each analyte. The analytical responses
of these samples were compared with those of the working
solutions.
As further tests to assess ion suppression, post-column infu-
sion experiments were done. By using an infusion pump (Model
11 plus, Harvard Apparatus, Holliston, MA), a continuous post-
column infusion (at 5 ␮l min−1 ) of standard solution (PMA,
BMA and MBMA 500 ␮g l−1 in 20 mM formic acid/MeOH 1:1,
v/v) was introduced into the analytical LC system through a T-
connector, during injection of an extract of urine (spiked with
ISs). Ion suppression of PMA, BMA and MBMA signal by the
urinary matrix was examined as “negative” chromatographic
peaks from the elevated baseline using six different urinary
samples.
2.9.4. Specificity
The specificity of the method was assessed by analysis of six
individual batches of control urine, each analyzed both unspiked
and spiked at the LOQ level. The peak heights for blank matrix
samples should not exceed 20% of peak heights at the LOQ
level and accuracy at the LOQ should be within 80–120% of the
nominal value.
2.9.5. Stability
Stability of the analytes and the ISs was investigated in stan-
dard solutions and in urinary matrix before and after sample
extraction, according to the FDA guidelines. Stability of the
stock and working solutions was evaluated for 8 h at room tem-
perature and under storage conditions (−20 ◦ C for 1 year and
+4 ◦ C for 1 week for stock solutions and working solutions,
respectively).
Stability in human urine was assessed in triplicate on three QC
samples (2, 20 and 50 ␮g l−1 ) after long-term storage (2 months
at −20 ◦ C), short-term storage (24 h at room temperature) and
after three freeze/thaw cycles, by comparison of the results with
those obtained from freshly prepared samples. Furthermore,
post-preparative stability was assessed in the final extract by
testing reproducibility in autosampler tray over a single batch
period (+15 ◦ C for 48 h).
3. Results and discussion
3.1. Analytical characteristics
3.1.1. Mass spectrometry
Fig. 1 shows the product ion mass spectra of the three ana-
lytes. For PMA, the product ion mass spectrum was recorded
in negative ion mode from the precursor ion m/z 238; the most
intensive fragment was detected at m/z 109 (Fig. 1a). Fig. 1b
shows the product ion mass spectrum of BMA: the signal at m/z
123 was monitored as the larger fragment of the deprotonated
molecular ion (m/z 252). For MBMA, the most abundant frag-
ment obtained from the precursor ion m/z 266 was detected at
m/z 137. PMAd-5 and BMAd-7 gave ions at m/z 243 and 114
and at m/z 259 and 130, respectively (data not shown). The main
fragments detected for all these compounds resulted from the
deprotonated ions by loss of CO2 and CH2 CH–NHCOCH3
(proposed fragmentations for PMA, BMA and MBMA are
shown in Fig. 1a–c, respectively). Our results on the nega-
tive ionization of PMA and BMA are consistent with those
previously reported [7,28]. Thus, for HPLC–MS/MS analysis
in MRM mode, m/z 238 → 109, 252 → 123, and 266 → 137
were selected as the most sensitive transitions for PMA, BMA
and MBMA, respectively. Transitions m/z 243 → 114 and m/z
259 → 130 were monitored for PMAd-5 and BMAd-7, respec-
tively.
 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122 119

3.1.2. Liquid chromatography

The elution gradient and influence of mobile phase were
investigated in order to optimize the analytical performances. As
reported in previous studies on the ESI− behavior of compounds
containing carboxylic acid groups, a mobile phase containing
formic acid in water/methanol gradient resulted to be opti-
mal [29]. We found that the response of acids increased when
the concentration of formic acid decreased. However, formic
acid increased analytes retention on the column and allowed to
resolve analyte peaks from interfering co-eluting matrix com-
ponents. A short analytical column and elution gradient with
20 mM formic acid and methanol were chosen as best compro-
mise between retention time and ionization of the three analytes
(data not shown). Under these chromatographic conditions, ana-
lytes and ISs were eluted in 10 min. Fig. 2 shows the MRM
chromatogram of a urinary calibration sample spiked with LOQ
concentrations of each analyte.

3.2. Method validation

3.2.1. Linearity, LOD and LOQ

The linearity of the calibration curves was determined over
the ranges 0.6–50.0 ␮g l−1 , 0.7–50.0 ␮g l−1 and 0.8–50.0 ␮g l−1
for PMA, BMA and MBMA, respectively. Each calibration
equation was fitted by the linear regression equation y = ax + b,
where y is the signal peak area ratio between the analyte
and its IS and x is the concentration of the spiked analyte.
Although isotopically labeled analogs would be the ideal ISs
in HPLC–ESI-MS analysis, isotopically labeled MAs are not
commercially available. Therefore, PMAd-5 and BMAd-7 were
synthesized to use as ISs for PMA and BMA, respectively.
BMAd-7 was also used to correct MBMA. In fact, we veri-
fied that, as compared to analysis without IS (data not shown),
the correction of MBMA signal by that of BMAd-7 increased
both the correlation coefficient of the calibration curve and the
accuracy of back-calculated concentrations, while guaranteeing
to meet the FDA requirements (see MBMA, Table 2). Fig. 2. Representative chromatogram of a calibration sample spiked with
For each analyte, the calibration curve showed a coefficient of 0.6 ␮g l−1 of PMA (A), 0.7 ␮g l−1 of BMA (B), 0.8 ␮g l−1 of MBMA (C) and
determination (r2 ) greater than 0.999. Concentrations were back 5 ␮g l−1 of each IS (D and E).
calculated from calibration curves for each calibration sample
(including LOQ): deviations from the nominal concentrations BMA and MBMA, respectively. Calibration parameters, LOD
(bias%) were between 0.1 and 12.1% for PMA, between 1.4 and and LOQ of the three analytes are summarized in Table 1. The
14.3% for BMA, and between 0.04 and 4.9% for MBMA. The LOQ of the method was determined for each analyte by divid-
R.S.D. values resulted to be <5.1, <2.7 and <21.0% for PMA, ing four times the S.D. of the ‘blank’ determination by the slope

Table 1
Calibration parameters obtained for PMA, BMA and MBMA against ISs from three different calibration curves prepared in triplicate and analyzed in different days,
within 2 weeks
PMA BMA MBMA

Range of calibration (␮g l−1 )a 0.6–50.0a 0.7–50.0a 0.8–50.0a

Slope (±S.D.) 0.283 (±0.016) 0.256 (±0.019) 0.200 (±0.015)
Intercept (±S.D.) 0.020 (±0.029) 0.027 (±0.042) 0.037 (±0.048)
Regression coefficient (r2 ) 0.9999 0.9998 0.9995
Limit of detection (␮g l−1 )b 0.30 0.35 0.40
Limit of quantification (␮g l−1 )b 0.60 0.70 0.80
a Assessed on seven concentration levels.
b Assessed by analysis of calibration samples (n = 6).
120 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122

Table 2
Precision and accuracy for PMA, BMA and MBMA in urinary QC samples
Nominal concentration (␮g l−1 ) Intra-day Inter-day

R.S.D. Bias% R.S.D. Bias%

PMA
2 9.0 −8.9 5.6 −5.7
20 3.9 0.1 6.2 2.9
50 5.0 −4.0 2.8 −2.0
Average 6.0 −4.3 4.9 −1.6
BMA
2 2.8 −4.4 14.0 2.0
20 4.7 8.8 6.0 8.2
50 2.2 10.8 2.7 5.8
Average 3.2 5.1 7.6 5.3
MBMA
Fig. 3. Chromatogram of an extracted urine sample (spiked with ISs) with post-
2 8.2 7.9 5.3 9.4
column infusion of 500 ␮g l−1 of PMA, BMA and MBMA.
20 4.1 14.2 4.1 14.2
50 1.7 14.9 3.8 11.6
in different batches of urine has been included as part of the
Average 4.7 12.3 4.4 11.7
validation procedure.
Number of replicates = 6. The experiments performed on six different urine samples
spiked after extraction showed a high-ion suppression (mean
analytes signal was 25.3% as compared to standard solutions).
of the calibration equation. Moreover, the accuracy of quantifi- These results are confirmed by post-column infusion experi-
cation at the LOQ level should be tested in six different urine ments, which indicated that the analytes peaks fell into an ion
lots and should be between 80 and 120% for all urines. Sev- suppression region (see Fig. 3).
eral LC–MS published methods reported slightly lower LOQ In the attempt to decrease ion suppression by increasing
values for PMA or BMA in urine. However, these values were analytes retention in column, several elution gradients were
obtained by analysis of a pool of urine and not from different tested. However, when the analytes retention time changed, the
urine samples [13,15–19]. Due to the unavailability of a urine region of maximum ion suppression moved accordingly (data
sample completely free of BMA, the determination of the LOQ not shown). This may be due to the fact that ion suppression of
for BMA presented some problems. An estimated LOQ value the urinary matrix in this region is caused by other MAs, which
for BMA in urine was thus calculated to be 0.7 ␮g l−1 . Never- are present in urine at high concentration levels. For the same
theless, the actual LOQ of BMA in urine may be lower than this reason, different extraction procedures performed using differ-
estimate and could be determined only in urine samples with ent SPE cartridges (Isolute® C18 and Isolute® Env+ ) did not
non-detectable amounts of BMA. decrease matrix effect (data not shown).
Several LC–MS analytical methods for the determination of
3.2.2. Precision, accuracy and recovery PMA or BMA have been published, but only few showed ion
Table 2 shows the intra-day and inter-day accuracy and preci- suppression data. Although obtained using a different clean-
sion values of the entire procedure for PMA, BMA and MBMA. up procedure, our results are consistent with those previously
For all analytes, accuracy was within 91.1 and 114.9% and published [7].
R.S.D. did not exceed 14.0%. This showed that each analyte The repeatability of the total procedure was evaluated ana-
met the generally accepted criteria for bioanalytical method lyzing six calibration curves constructed in six different lots of
validation at all QC concentration levels. urine, instead that in a single (pooled) urine sample. Despite the
As regards recovery obtained from the SPE procedure, the high suppression due to urinary matrix, the slopes of calibra-
mean values were 82 (±4.4) % for PMA, 71.2 (±7.8) % for tion curves measured with six different lots of urine were highly
BMA and 78.3 (±11.8) % for MBMA. precise (R.S.D. < 8%), and an identical susceptibility of analytes
and ISs to matrix effect may be inferred. This, together with satis-
3.2.3. Matrix effect and ion suppression fying inter-day precision and accuracy values obtained for LOQ
Although matrix-induced alterations (suppression or concentration levels from the six lots of urine was indicative for
enhancement) of the ESI-MS/MS signals may critically impair the absence of matrix-caused quantification errors.
the reliability of a method, little attention is often paid to
this topic during method validation. In particular, previously 3.2.4. Specificity
published methods for the determination of MAs in urine did The method showed good specificity, meeting the accep-
not test matrix effect and ion suppression [13,15–20]. Due to tance criteria of the FDA. No interfering peaks were detected
the complexity of biological fluids and to the high inter-subject in the ‘blank’ samples at the mass transitions chosen for PMA,
variability of urine in particular, assessment of matrix effects MBMA and the ISs. The signal detected for BMA in all unspiked
 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122
Table 3
Results of HPLC–MS/MS analysis in urine samples of 354 traffic wardens
% of quantifiable Urinary concentration
samples mean ± S.D. (␮g/gcreatinine )
PMA 27 1.76 ± 1.58
BMA 100 12.84 ± 25.71
MBMA 23 3.98 ± 7.19
tested lots (signal to noise ratio larger than 3:1) corresponds to
background levels of BMA in urine. The accuracy of the six
individual urinary samples spiked at LOQ resulted within the
parameters indicated in the guidelines (80–120% of the nominal
value).
3.2.5. Stability
All the stability experiments performed met the FDA require-
ments: the deviation from the initial concentrations of the
analytes and ISs was <6% in the standard solutions. No sig-
nificant changes in concentrations (≤14%) were observed in
urinary QCs.
3.3. Application of the method: MAs determination in a
group of traffic wardens
The validated analytical LC–MS/MS method was applied to
measure the urinary levels of PMA, BMA and MBMA in 354
subjects (225 men, 129 women; 105 smokers, 249 non-smokers)
involved in traffic control in Bologna (Italy). Spot urine sam-
ples were collected at the end of an 8 h work shift during the
period April–November 2006. Since the concentrations of these
metabolites, which are excreted by diffusion, are dependent on
urine output, correction for creatinine concentration is neces-
sary. Urinary creatinine was determined by the Jaffé method. As
shown in Table 3, all samples showed a measurable concentra-
tion of BMA, while PMA and MBMA resulted quantifiable only
in 27% and 23% of the analyzed urines, respectively. No sample
exceeded the BEI value proposed for PMA (25 ␮g/gcreatinine ) by
the American Conference of Governmental Industrial Hygien-
ists (ACGIH) [11].
No statistically significant differences were found between
male and female subjects in the urinary excretion of these
metabolites (all p > 0.05). The difference in urinary concen-
tration between smokers and non-smokers was not statistically
significant for BMA and MBMA, whereas it was statistically sig-
nificant (p < 0.05) for PMA (smokers: 1.97 ± 1.67 ␮g/gcreatinine ;
non-smokers: 0.98 ± 0.87 ␮g/gcreatinine ). Moreover, it is interest-
ing to note that 80% of the samples showing measurable amounts
of PMA were urines obtained from smokers. This is not surpris-
ing, since it is common knowledge that tobacco smoke represents
an important source of benzene. In fact, several studies reported
higher concentration of PMA in smokers than in non-smokers
[7,30]. Furthermore, previous studies demonstrated that traffic
wardens are exposed to relatively low levels of benzene and
identified tobacco smoke as the main source of benzene among
these workers [30,31]. Our results are in line with these data.
Moreover, the PMA levels reported in our study are very similar
121
to those obtained by Bono et al. [30] in a group of 206 traffic
wardens.
4. Conclusions
To our knowledge, this is the first fully validated
HPLC–MS/MS method for the simultaneous determination of
PMA, BMA and MBMA in human urine. The method meets
all the FDA acceptance criteria over the following ranges:
0.6–50.0 ␮g l−1 , 0.7–50.0 ␮g l−1 and 0.8–50.0 ␮g l−1 for PMA,
BMA and MBMA, respectively. Moreover, the high sensitiv-
ity and sample throughput make the method suitable for the
biological monitoring both of occupational and environmental
co-exposure to BTX.
Acknowledgements
We are grateful to Dr. Chiara Scardoni for scientific editing.
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